DE4227769A1 - Bacterial strains for mercury reduction and processes for their production - Google Patents

Description

The biological basis of mercury (Hg) toxicity is the ability of Hg in its divalent cationic Form [Hg (II)], sulfhydryl, thioether and imidazole groups too bind and thereby inactivate enzymes (26). It is therefore generally recognized that Hg (II) is the strongest is toxic ionic form of the metal. Organic Mercury species, both alkyl and aryl derivatives, can accumulate in the tissues of higher organisms where they cause systemic diseases (17). In spite of the extreme biotoxicity of Hg (II) and its organic However, derivatives are estimated to produce 11,000 tons a year Hg and Hg-containing compounds mobilized by human hands releases ties into the biosphere. So it can be say that mercury pollution is a serious one Pose a risk to the environment.

Since the total amount of mercury present is final and unchanged can be remedied only with regard to mercury pollution can be achieved in that the ionic form in a less toxic species is transferred and / or that they are separated ideally in one for further use in the Circulatory traceable form. Current Hg-Beseiti technologies use adsorption matrices to remove mercury silver compounds from industrial exhaust gases or waste water to remove. However, this procedure makes it more solid Secondary waste generated, and bound Hg is for a white no longer available. The procedure points continue to suffer from the disadvantage that it is relatively unselective; waste components other than Hg are also adsorbed.  

An alternative strategy to eliminate that currently is investigated, the conversion of Hg (II) into the inert, volatile elementary form [Hg (0)], namely under Use of the mechanism of bacterial mercury resistance that on the reduction of Hg (II) to Hg (0) through the activity a cytosolic mercury reductase (8, 36). Such bacterial reduction systems could either be in place and place used for disposal because volatile made Hg (0) both less toxic and less bio is available, or within bioreactors which the reduced Hg (0) can be recovered if necessary could.

The specific bacterial mercury resistance (Hg ^r ) involves mercury reductase on the one hand and, on the other hand, since Hg (II) also inactivates membrane proteins, an Hg-specific transport system is involved, which transports the membrane and other cellular components by transporting the Hg of protects the cell surface from intracellular mercury reductase (24).

Hg ^r with a narrower spectrum of activity confers resistance only to mercury ions. In addition to the transport and reduct enzymes, an organomercury lyase is involved in the mercury resistance with a broad spectrum of activity, which protonolytically cleaves carbon-Hg bonds (2, 36, 41). The Hg ^r systems with a broad spectrum of activity therefore confer resistance to organo mercury compounds through the successive activities of lyase and reductase.

The Hg ^r mechanism has been found to a large extent in bacteria for both a narrower and a broad spectrum of activity. The Hg ^r structural genes are encoded in a single operon that is primarily regulated by the merR gene product, which in turn is transcribed adjacent to the merTPABD operon, but in the reverse direction thereof. The merT and merP gene products are involved in the uptake and transport of Hg (II), merA specifies the mercury reductase, and merB codes for the organo mercury lyase (39 contains an overview). merD, the most distant promoter gene that has been identified, has been implicated in a transcriptional function of co-regulation (22, 29).

It is an object of the invention to verbes bacterial strains Serious mercury detoxification properties are available too put.

This problem was solved in that tribes of Pseudomo nas putida are made available, which as a result of con statutory hyperexpression of the merTPAB genes of such verbes Serte have mercury detoxification properties. Erfin According to the invention, organisms are also provided in which the ability to increase Hg at high concentrations To reduce rates at the genetic level with a benzene Catabolism is combined. The corresponding Pseudomonas putida derivatives are able to match the chemically unequal Components of an organomercury compound, namely Phenylmercury acetate (PMA), in their metallic and disassemble aromatic components and separate each detoxify. The characterization of these isolates shows that for the treatment of mercury-containing impurities are suitable.

The strains provided are characterized in that she

• a) are able to make mercury (II) ions from mercury to reduce salts and organo mercury compounds,
• b) high resistance to mercury compounds point,
• c) an expression apparatus for the mercury detoxifiers Own proteins that are completely independent of the surrounding ones Mercury concentration is  
• d) genetically stable insertions of overexpressing Genes for mercury resistance in the host chromosome point, and
• e) optionally break down additional toluene and benzene groups can.

These strains detoxify organic mercury compounds like also mercury salts not only at very high, but also at very low mercury concentrations, which tere normally, e.g. B. in the wild types, not for induc tion of the synthesis of mercury detoxifying proteins pass. Those strains that also have benzene and toluene dismantle groups, these groups essentially set ultimately to carbon dioxide and water regardless of whether the benzene and / or toluene as an organic group in one Mercury compound or as a connection in itself in the order of the degrading organism.

Pseudomonas putida is preferred for generating the mutants wisely mutagenized with a mini transposon that merTPAB contains, i.e. structural genes that contain the mercury specify resistance. The transposon should preferably be one or have more than one of the following:

• i) A very short ending sequence (e.g. 19 bp).
• ii) Transpose only once into the acceptor or recipient; the transposed DNA should be stably integrated into the host, do not hike, do not double and do not go through Action of one of its own components be cut.
• iii) The transposon construct should not have mer regulatory genes exhibit.

If such a transposon is downstream (in the 3′-direction) to those regions of the host DNA that are high Cause transcription rates, so the genes because of the short distance to the strong host promoters by this be expressed as the RNA polymerase attached to the promoter binds and through the short intermediate piece the mer gene  reads. Due to the high transcription rate, a high Copy number of the RNA transcript generated for the proteins encoded for the (organo) mercury resistance and Ent poisoning are responsible.

Following the mating of the cells, these are selectively some strains isolated, the merTPAB constitutively hyperexpri lubricate what gives them the ability to increase Hg in rates under conditions of high mercury concentrations from one split off organic group and the released Hg (II) in the less toxic and biologically unusable form To transfer [Hg (0)].

In a second embodiment, these properties genetically linked to a benzene / toluene catabolism, so that microorganisms are provided which not just the metal component of phenylmercury acetate (PMA) can reduce, but also complete degradation of its aromatic group. These isolates are therefore capable of making PMA the only carbon and energy source to use.

The purposeful adjustment of metabolic processes through "genetic engineering" opens up the possibility of improved Preparations for detoxification from environmental pollution put. Different strategies have been used to to achieve this goal. It succeeded in intermediate products of catabolism by using alternative catabolic Channel pathways, as well as "not allowed" steps in catabolism by sequential mutation selection to eli minimize, causing the expansion of the pathway substrate profile is made possible (32, 34, 42). The present invention shows another approach: The enzymatic cleavage of chemically unequal units of a compound class, näm of an organometallic, into the individual components, Hg (II) and benzene, and the connection of pathways to each of the detoxified formed components separately.  

The invention accordingly addresses both consti tutively hyperexpressing mercury detox determinants for the production of highly resistant to (organo) mercury Strains of bacteria that target their compounds under increased Hg Concentrations in higher rates can detoxify as well on linking these properties with the degradation of Benzene, so that mercury-benzene derivatives completely abge can be built.

While changes in regulation in the synthesis of catabolic enzymes previously focused on modifying the effector properties of certain regulator proteins (32, 42), the constitutive hyperexpression of Hg ^r with a broad spectrum of activity is, according to the invention, eliminated by eliminating the regulatory control for mer and that Targeted mutagenization of P. putida hosts achieved with a MerTPAB-containing mini Tn5 element. It has been assumed that the transcription of mer genes is triggered by promoters of the exogenous host and is read by abbreviated IS50 sequences.

The plasmid pHG106 contains a 4.2 kB HindIII frag ment that the merTPAB DNA for the construction of the by pUTHg encoded mini-Tn5 (11, 12). The upstream HindIII site located for the construction of both pHG106 and pUTHg could be used, however a deletion of the -35 and primary -10 sequences of the have merTPABD promoters (Pmer) (16, 28). The one for merTPAB DNA encoding in pUTHg certainly shows both deletion of the merR and the merD regulator gene on (11, 12), and you could also use elements of the primary transcription con trolls are missing.

Testing a large number of P. putida mini-Tn5 acceptors after conjugation showed that there was a wide range of PMA resistance among the isolates tested. The further characterization of selected, screened isolates also showed diverging values for Hg ^r ( FIG. 1), which correspond to different values for the mercury reductase activity (Table 2, FIG. 2). Southern blot analysis showed that transposition events had indeed occurred and that a single copy of merTPAB was present in each of the isolates tested ( Figure 4). Therefore, the comparison of different P. putida :: mer strains shows different values of the merTPAB expression, which is probably due to different promoter activities, although a thorough characterization of the transcription parameters in these isolates is still pending. The data presented here are therefore in line with the fact that the transcription of merTPAB in the P. putida :: mer strains generated and analyzed is at least partially carried out by exogenous promoters of the host.

The selection of hyperexpressing P. putida :: mer derivatives was achieved by screening for PMA, an original protocol that suppresses the false positive background that occurred when screening for HgCl ₂ . Mer hyperexpressing P. putida progeny proved to be significantly stronger Hg ^r / PMA ^r , a finding that not only enabled their selection but is also new, since the same effects could not be produced in E. coli (23, 25 , 31).

The inventors examined approximately 2000 with mini-Tn5-mer mutagenized derivatives of P. putida KT2442 and F1. The following strains were in the German collection for Microorganisms deposited:

P. putida KT 2442:: mer-73 DSM 7 209
P. putida F1:: mer-1-1 DSM 7 208

With the given number of isolates tested, the probability that the highest achievable value for the PMA ^{r should have been} found under the conditions used. If higher values of resistance are found in vivo due to the limitations of Hg (II) transport, these may have been defined in P. putida by the method described here. An alternative attempt to achieve even higher Hg (II) reduction in vivo could be to bring the transport determinants, merTP, under defined, but separate from merAB regulatory control. merAB codes for the (organo) mercury transforming functions.

Constitutive hyperexpression of merTPAB apparently produces immediate Hg ^r , even under toxic concentrations of Hg (II), and no effects of mer hyperexpression on the growth rates of the cultures were found ( Fig. 3). By combining the constitutive hyperexpression of merTPAD with the capacity of P. putida F1 to degrade benzene, both growth and detoxification of PMA was achieved when it was used as the sole source of carbon and energy.

The isolates of P. putida :: mer have beneficial properties for the detoxification of mercury containing waste both in containers and in the open Environment. Since the solution strategy of the present Er is based on enzymatic reduction, it is specific for (formed) Hg (II) and avoids the unselective eigen currently used adsorptive systems.

The strains described here are able to Too awake in the presence of highly concentrated mercury compounds sen and express this ability constitutively; the Cell death in highly contaminated waste before complete Induction of expression is prevented. Furthermore, is under Conditions in which the Hg (II) concentrations in the Environment are below toxicity and are too low, to induce Hg (II) reduction in established populations ren, constitutive bio-detoxification is also an advantage.

P. putida is a naturally occurring, soil-colonizing organism that is almost ubiquitously distributed. This is why P. putida :: mer strains should pose fewer risks in maintaining the natural ecological balance if they are deliberately released as a means of neutralizing (organo) mercury. Genetic analysis also shows that a single copy of the mer operon is stably built into the host chromosome ( Fig. 4); the risk of transmission of merTPAB to organisms naturally occurring in the corresponding environment should therefore be significantly reduced.

P. putida :: mer isolates detoxify both Hg salts and Or gano mercury compounds, and P. putida F1 :: mer derivative linge are able to use benzene and toluene mercury derivatives to use only carbon source. This makes it possible speed, mixtures of mercury-containing compounds as well as benzene and detoxify toluene, which in turn is toxic. The Need to add nutrients to the system from outside, could also be superfluous while a Toxicity to the cell due to an accumulation of aromati end products is avoided.

By manipulating the naturally occurring bacterial Systems for the detoxification of mercury compounds will be improved ways and means of treating Mercury-containing waste streams and contaminated locations made available.

The ability to transform Hg is by means of Atomic absorption spectroscopy, on-line, has been determined. One is in Compared to P. putida KT2442 :: Tn501-132, improved 10-fold Sales speed has been determined.

The invention is intended in the following with reference to figures, tables and examples are explained in more detail.

The figures show in detail:

In Fig. 1, the maximum resistance of P. putida :: mer isolates against HgCl ₂ and PMA is plotted. These are the highest concentrations of PMA in solid (∎) or liquid () and of HgCl ₂ in solid () or liquid () LP121 medium, at which growth of the P. putida :: mer strains still occurs was possible. 10 or 15 µg / ml PMA in liquid or solid LP121 medium inhibited the growth of P. putida KT2442 :: Tn501-132 and the parental strains P. putida KT2442 and F1. The growth of the parental strains was also inhibited by HgCl ₂ in concentrations of 6 or 35 µg / ml in liquid or solid LP121 medium.

Fig. 2 shows the correlation of PMA or HgCl ₂ shows -resistance with the merTPAD expression. The mean mercury reductase activity (in the presence of or without Hg, Table 2) of 22 isolates examined as a function of their maximum PMA resistance, observed in liquid (○) or solid (⚫) LP121 medium, resp shown as a function of their maximum mercury resistance in liquid () or solid LP121 medium (∎). Square equations for the graphs with the corresponding correlation coefficients (R ² ) are given above each graph. The F-value, a statistical determinant for the adaptation of the data to the calculated graphs, was 8.74, 3.05, 2.45, 1 for the data of the graphs A, B, C, D and E, respectively , 52 and 6.53 respectively. Values greater than 4.32 for F (representations A, B, C and D) or 3.96 (representation E) show a significant (95% certainty) correlation between the data and the function shown.

Figure 3 shows the growth of P. putida strains in the presence of HgCl ₂ . LP121 media with 6 µg / ml HgCl ₂ (∎), with 6 µg / ml HgCl ₂ , added at A ₆₀₀ = 0.5 (▲), or without the addition of HgCl ₂ (○) were grown with a culture in stationary Inoculated phase (1: 100) and incubated at 30 ° C. P. putida :: Tn501-132 was pre-induced with 10 µM HgCl ₂ in the midlog phase for 2 hours before the addition of HgCl ₂ .

Figure 4 shows a Southern hybridization analysis of P. putida :: mer strains. The blots were probed with a labeled fragment (3.2 kB) derived from pUTHg and containing merTPAB. The traces of Fig. (A) show P. putida :: mer KT2442 strains: 1: P. putida :: mer KT2442 (SfiI); 2: -67 (SfiI); 3: -73 (SfiI); 4: -2-4 (SfiI); 5: -2-49 (SfiI); 6: -2-121 (SfiI); 7: pUTHg (SfiI); 8. -67 (EcoRI); 9: -73 (EcoRI); 10: -2-4 (EcoRI). The traces of Fig. (B) show P. putida :: mer F1 strains: 1: P. putida :: mer F1 (SfiI); 2: -1-1 (SfiI); 3: -1-7 (SfiI); 4: pUTHg (SfiI); 5: -1-22 (SfiI); 6: -10 (SfiI); 7: -13 (SfiI); 8: putida :: mer KT2442 :: Tn501-132 (AvaI); 9: pUTHg (EcoRI).

Table 2

Mercury reductase activities from HgCl ₂ induced or uninduced P. putida :: mer cultures

Table 3

Growth and catechol-2,3-dioxygenase activity of P. putida F1 :: mer isolates on minimal media with PMA as the only carbon source

Examples Example 1. Materials and methods a) Bacterial strains, plasmids and growth media

The bacterial strains and plasmids used in the present invention are shown in Table 1. L medium contained 1% tryptone (Difco), 0.5% yeast extract (Difco) and 0.5% NaCl. M9 and M63 minimal media (19) were each supplemented with 0.2% citrate as a carbon source. 0.001% Pseudomonas stanim salts ( 1 ) additionally supplemented the minimal M9 medium; 0.1% casamino acids (Difco) were added to the M63 minimal medium. Minimal salt medium with a low phosphate content (100 μM) (LP121) was prepared as previously described (14). The media were solidified with 1.5% agar (Difco). Antibiotics were used in the following concentrations (per ml) in selection media: rifampicin (Rif): 50 µg; Kanamycin (Km): 30 µg; Ampicillin (Ap): 50 µg; Carbenicillin (Cb): 500 µg; Piperacillin (Pc): 30 µg. Hg (II), in the form of HgCl ₂ (Sigma) from a stock solution of 10 mg / ml, was added to the pre-cooled media in the stated concentrations. Concentrated stock solutions of phenylmercury acetate (PMA) (Sigma) (1 mg / ml) were shaken vigorously at 30 ° C for 2 hours before being added to the media at the concentrations indicated.

b) Mobilization and transposition

The transposon-containing plasmid was transferred from Escherichia coli SM10 lambda pir (pUT-Hg) by mobilization with a biparental filter-mating technique into either P. putida KT2442 or P. putida F1 recipients (6). Individual cultures of the donors and the acceptors were grown to the stationary phase at 30 ° C. in L medium supplemented with selective antibiotics. 0.4 ml of the acceptor strain and 0.1 ml of each donor were mixed in 5 ml of L medium and then filtered (0.2 u Swinnex). The filters were placed cell-side up on the surface of L medium plates and incubated at 37 ° C for 9 hours. The conjugation products were then resuspended in 5 ml of 0.85% saline and suitable dilutions were plated on selective media. M9 medium supplemented with Rif and 6 µg / ml HgCl ₂ was used to select for pFRC37p mobilization and Tn501 transposition in P. putida KT2442. M63 medium with Rif and 1.5 µg / ml HgCl ₂ was used to select for pUTHg mobilization and mini-Tn5 transposition in P. putida KT2442. M63 medium, supplemented only by 1.5 µg / ml HgCl ₂ , was used for the selection for the mobilization of pUTHg and the mini-Tn5 transposition in P. putida F1. Selective plates were incubated at 30 ° C. The exconjugants were either on M9 medium with Rif and 6 µg / ml HgCl ₂ on the pairing C600 (pFRC37p) P. putida KT2442 or on M63 medium, supplemented by 10 µg / ml PMA, on the acceptors of the pUT-Hg -Pairing results tested. The screened conjugation products were also examined to determine whether, as far as P. putida KT2442 was concerned, they were resistant to Rif (Rif ^r ) or whether they were sensitive to Cb, Pc and Km (Cb ^s , Pc ^s or Km ^s ).

c) Determination of increased values of the Hg resistance.

The conjugation products of the P. putida strains were replica-plated from L medium plates with 10 µg / ml PMA to L medium plates with 50 to 250 µg / ml PMA. Those strains that showed growth of isolated colonies at elevated PMA concentrations were further examined for the growth of isolated colonies on LP121 plates containing either 1.5 to 60 µg / ml HgCl ₂ or 10 to 75 µg / ml PMA. The strains of P. putida were examined for the upper limits of Hg ^r in liquid medium by growing the cultures of isolated conjugation products at 30 ° C. in LP121 medium until the mid-log phase (A ₆₀₀ = 0.5) whereupon HgCl ₂ or PMA was added depending on the final concentration (alternatively, the pFRC37p exconjugants at A ₆₀₀ = 0.5 were induced with 0.8 µg / ml HgCl ₂ and then preincubated for two hours, after which HgCl ₂ until Final concentration was added). The A ₆₀₀ was determined after about 4 hours of growth in HgCl ₂ or PMA and compared with the A ₆₀₀ parallel cultures which had been grown under the same conditions but without the addition of HgCl ₂ or PMA.

d) Determination of the growth curves

Stationary cultures of P. putida strains were inoculated in LP121 minimal medium (1: 100). Parallel cultures that were at 30 ° C without HgCl ₂ or with an initial concentration of 6 µg / ml HgCl ₂ or with an addition of 6 µg / ml HgCl ₂ after reaching the mid-log phase (Tn501-containing strain were for one hour pre-induced with 2.7 µg / ml HgCl ₂ before 6 µg / ml were added to the midlog) were monitored at regular intervals by determining the A ₆₀₀ . Counting plates with live cultures was used to ensure the results.

e) Mercury reductase determinations

The cells were grown at 30 ° C in LP121 cultures from 50 ml to A ₆₀₀ = 0.5 where they were either allowed to continue growing without the addition of HgCl ₂ or where HgCl _{2 was added} at the highest concentration, the growth still (if they were conjugation products (ex-conjugant strains) of pUTHg), or where they were induced with 0.8 µg / ml HgCl ₂ for 2 hours (strain containing Tn501) and then HgCl _{2 was added} to 6 µg / ml . The cultures were grown to a final value of A ₆₀₀ = 1.5 and then harvested by centrifugation (10,500 x g, 10 min., 4 ° C.), once with 20 ml of cold 100 mM NaPO ₄ (pH 7.5 ), 0.5 mM EDTA (Fluka), 0.1% β-mercaptoethanol (Sigma) and resuspended in 1 ml of the same buffer. The cells were lysed with ultrasound (50W, 2 min., B. Braun Labsonic U) and the insoluble fraction was centrifuged off (27,000 × g, 4 ° C., 30 min.). The supernatant was tested for mercury reductase using a modification of the method by Fox and Walsh (8) and Schottel (36). For this purpose it should be briefly stated that 0.2 ml of crude extract was added to a test mixture, the final concentrations of 50 mM NaPO ₄ (pH 7.5), 200 μM NADPH (Sigma), 1 mM β-mercaptoethanol and 100 μM HgCl ₂ in one Contained a total reaction volume of 3 ml. The reactions were carried out at 25 ° C for 4 minutes and during this time the oxidation of NADPH was monitored spectrophotometrically at 340 nm; the background values of the NADPH oxidation were determined by observing parallel reactions without HgCl ₂ . The protein concentrations were determined using the BCA determination (Pierce), carried out according to the recommendations of the manufacturer.

f) Southern blot

Genomic DNA was isolated by the method of Richards (33); Plasmid DNA was isolated by alkaline lysis and purified by CsCl equilibrium centrifugation (35). 10 µg of genomic DNA was digested with either Aval or SfiI or EcorI, and 0.1 µg of the plasmid DNA was cut with SfiI (Boehringer Mannheim) according to the manufacturer's instructions. Digested DNA samples were run over a 0.7% agarose plate gel (35). The gels were blotted on Biodyne "B" membranes (Pall Biosupport) and the membranes were hybridized with labeled DNA probes according to the manufacturer's suggestions. The DNA fragments used as probes were isolated from agarose plate gels using Gene Clean (Bio 101, Inc.,); Fragment and plasmid DNA used as a probe were labeled in vitro with [alpha- ³² P] -dCTP (Amersham), using random primer extension ("multiprime DNA labeling system") , Amersham).

Example 2. Construction, selection and screening of P. putida derivatives with high resistance to PMA (PMA ^r )

Theoretically, the hyperexpression of determinants could broad spectrum of activity to increased resistance values against about organo mercury compounds and at the same time an increased the detoxification properties of the resulting strains lead to (organo) mercury compounds.

In order to achieve an overproduction of the mer gene products, According to the invention, the plasmid pUTHg is used, which is a Repli kon with narrow host specificity and for the merTPAB-mercury silver resistance with a broad spectrum of activity, from pDU1358 originating, coded (11). The pUTHg-merTPAB genes are derived from the inverted repeat sequences flanked by Tn5 that are reduced to 19 base pairs (bp). The transposase gene (tnp *) is on pUTHg regarding the transposing element of Tn5 arranged externally, and merR and merD, which for mer- Regulating regulatory proteins were derived from the elimi construct niert (see below, 11, 12).

Since previous studies have shown that an increase in mer gene dose in Escherichia coli not equally increased mercury resistance (23, 25) the two strains of P. putida as acceptors for pUTHg in biparental matings selected. P. putida F1 is one in the Naturally occurring, soil-isolating isolate (10) rend P. putida KT 2442 a rifampicin resistant Is the descendant of an isolated soil organism (18). Floor isolates were primarily used because of their potential uses for the restoration of floors contaminated with mercury and water used. Both strains are already in the Ver has been characterized, and F1 includes one through its chromosomes coded pathway for the catabolis mus of benzene (10). So the benzene breakdown could be in this Organism coupled with the detoxification of mercury become.  

After the "mating", ie the "pairing" of the acceptors P. putida KT2442 or F1 with the appropriate donor strain below Conjugation conditions, it was found that the Transposition frequencies for the merTPAB genes in more meaningful Way with previously observed (12). To white easier screenings and resistance to Securing organo mercury compounds was 1025 Iso late from the conjugation of P. putida KT2442 and 800 from the from P. putida F1 to L medium supplemented with 10 µg / ml PMA transfer.

Since it was assumed that the mini-Tn5-merTPAB transposition was generally undirected with respect to the target sites, it was concluded that inserts located downstream of proximal host promoters could lead to an increase in mer expression, whereby Hg and PMA resistance (Hg ^r PMA ^r ) should increase at the same time. Therefore, the further selection of the acceptor isolates with increased Hg ^r / PMA ^{r was} carried out by screening these products of the conjugation of P. putida KT2442 and F1 on mercury (II) -containing L medium. Since it was found that at high cell densities the Hg-sensitive (Hg ^s ) strains often grow on media containing HgCl ₂ , the less volatile phenylmercury acetate (from 50 to 250 µg / ml) was preferably added to the L media. This method avoids the selection of false positive clones; the resistant phenotypes can be clearly distinguished from the sensitive ones.

Different growth of P. putida KT2442 · pUTHg-Exkon jugants were observed on media containing 50 to 80 µg / ml PMA contained while the P. putida contained F1 · pUTHg isolates different growth on media showed 50 to 250 µg / ml PMA included. Above these upper limits of the PMA-Konzen none of the isolates tested grew. Separate representative conjugation product strains (20 P. putida KT2442 · pUTHg and 20 P. putida F1 · pUTHg), the growth isolated colonies on PMA-containing L medium selected for further investigation.  

These 40 exconjugant strains were tested on their ability tested to awake on L media containing Cb, Pc or Km sen, and all the tribes proved to be sensitive to it all three antibiotics (P putida was also found KT2442 derivatives as resistant to Rif), whereby it was ensured that there was no replication or integration one of the donor plasmids takes place in the investigated acceptors had found. So it could be shown that all Strains that have undergone further testing are one Transposition of mini-Tn5 into the host chromosome (:: mer) un had been thrown.

Example 3. Determination of the upper limits of resistance to HgCl ₂ and PMA in minimal media

The maximum levels of HgCl ₂ and PMA that still allowed growth in both solid and liquid LP121 were determined for the 40 P. putida :: mer strains previously examined. For these experiments, P. putida KT2442 :: Tn501-132, which contains a stable insertion of Tn501 (3, 38) coding for a low-spectrum mer operon, was constructed by mobilizing pFRC37p (see Example 1). P. putida KT2442 :: Tn501-132 provided a comparison value for prototypical Hg ^r and expression by a native mer promoter.

P. putida :: mer strains that showed growth of isolated colonies on LP121 agar with HgCl ₂ or PMA were classified as resistant to the added concentration of mercury compound. They were then tested in the same way at higher concentrations of HgCl ₂ and PMA until the values were reached that inhibited the growth of isolated colonies.

The maximum values of resistance in liquid LP121 media were determined by incubating cultures under aerobic conditions until the midlog growth phase had been reached (A ₆₀₀ = 0.5) and the mercury compound had been added at this point. The spectroscopic absorbency was determined for each culture again after four hours of continuous growth. The differences in optical densities were calculated and compared with the differences in absorbance of parallel, untreated cultures measured at the same time. Since sensitivity to HgCl ₂ or PMA at a given concentration led to a negligible increase in growth (data not shown), resistance was defined as approximately the optical density of the untreated control. Representative results of these experiments are shown in FIG. 1.

85% of the 20 P. putida KT2442 :: mer isolates tested showed a 12.5-37.5% increase in Hg ^r compared to P. putida KT2442 :: Tn501-132 when on solid LP121 -Media were grown. However, only 30% of the same strains show an increase in Hg ^r when grown in liquid LP121; The increase in these cases was 28.5% above the values of P. putida KT2442 :: Tn501-132. However, those P. putida KT2442 :: mer strains showed the greatest demonstrable resistance to growth in liquid that showed the highest Hg ^r on solid media. When these 20 P. putida KT2442 :: mer isolates were tested on PMA-containing LP121 media, they showed higher resistance over a shorter range of PMA concentrations on solid media than when grown in liquid media.

In general, the 20 P. putida F1 :: mer strains showed a larger Hg ^r than the P. putida KT2442 :: mer examined. 50% had a 14.3-85.7% increase in Hg ^r on solid or liquid LP121 media compared to P. putida KT2442 :: Tn501-132. In contrast, the upper limits for PMA ^r between the mer offspring of P. putida KT2442 and F1 were comparable ( FIG. 1).

DNA sequences distal to the merA genes of Tn501 and the plasmids R100 and pDU1358 are important for the Hg ^r and have a high degree of homology (4, 11, 13, 22, 25, 29). Deletion of these sequences, which code for at least two open reading frames (merD and urf1), reduces the maximum Hg ^r by 20% (4, 22, 25, 29), which is associated with a decrease in the induction of Hg volatilization (4 ) and correlated with a simultaneous increase in mer transcription (22, 29). It could be shown that the merD protein binds specifically to the mer promoter (P _mer ) (22). Therefore, it has been suggested that MerD acts as a coropressor of merTPABD expression (22, 29).

The EcoRV site distal to the promoter, which was used to isolate the merTPAB-containing fragment for the construction of pUTHg (12), is located upstream from merD (11). The broad-spectrum mercury resistance encoded by mini-Tn5 therefore shows a deletion of merD. Despite the loss of merD, however, it was found that some of the P. putida :: mer descendants examined here have a greater maximum Hg (II) / PMA resistance than P. putida, which harbors wild-type Tn501 ( FIG. 1). The fact that the resistance of some mini-Tn5 isolates was far higher than that previously observed in merD mutants, namely 60 µM (22), confirms that the mer-TPAB transcription in these isolates depends on strong exogenous host promoters their respective chromosomal insertion targets.

Example 4. mer gene expression in P. putida :: mer isolates

Since the enzymatic activity of the merA gene product, the mercury reductase, can be determined from crude cell extracts (36), this was chosen as a measure of the gene expression of mer ge. Mercury reductase determinations were carried out on extracts of 12 representative P. putida KT2442 :: mer and 11 P. putida F1 :: mer isolates, which had been grown with and without HgCl ₂ in LP121 media. P. putida KT2442 :: Tn501-132 was also examined after the strain was induced in LP121 medium with HgCl ₂ to provide the comparative value for mer expression derived from the native Tn501-encoded mer promoter ( 16, 21, 28). The results of these experiments are shown in Table 2.

Expression of merA in P. putida KT2442 :: Tn501-132 was not recognizable without the addition of HgCl ₂ . As previously found in studies with E. coli hosts, Tn501-encoded merA expression with Hg (II) can be induced, as can the mer operon of plasmid R100 (39). The Hg (II) inducibility in these systems is caused by the activity of the MerR protein, which in the presence of Hg (II) acts as a positive regulator for merTPAD expression, but in its absence acts as a negative regulator (7, 16, 25, 30).

However, it has been reported that the wild type Tn501 encoded mer activity in Pseudomonas aeruginosa is almost constitutive; in the presence or absence of Hg (II), only a 30% difference in MerA activity was observed (4). However, it has been shown in the present case that the expression of the Tn501 operon in P. putida is H-dependent: no mercury reductase activity was found in cells not previously exposed to Hg (II) (Table 2), and uninduced cultures were Hg ^s (see below, Fig. 3).

In contrast to the inducibility shown in the P. putida strain containing wild-type Tn501, all 23 P. putida :: mer isolates tested had approximately the same values for the mercury reductase activity, regardless of the addition or absence of HgCl ₂ . Therefore, merTPAB expression is constitutive in these strains; the addition of Hg (II) is not necessary for merTPAB expression (Table 2).

The plasmid pHG106, the source of pDU1358-merTPAB-DNA for the construction of MiniTn5 lacks the merR gene (11, 12).  

merR mutants of Tn501 and plasmid R100 normally show microconstitutive merTPAD expression (ie 10 times above the normal repressed value but 100 times below the normal induced values) and they are phenotypic Hg ^s (25, 28). The wild-type pDU1358-mer operon (from which the mini-Tn5 used here was derivatized), like Tn501 and R100, is also inducible, but if it has a deletion of merR, it gives E. coli Hg ^r (11). The constitutive mer expression, which can be seen in the P. putida :: mer isolates examined here, is therefore probably a consequence of the lack of a merR determinant.

The value of constitutive mercury reductase activity, which the examined P. putida :: mer isolates had, among differed significantly from the induced merA Activity exhibited by P. putida KT2442 :: Tn501-132. P. putida KT2442 :: mer isolates showed up to 4.2 times Increase (isolate 73), and the strains P. putida F1 :: mer showed a maximum of 3.3 times (isolate 1-1) higher reductase activity as the strain containing Tn501. The deletion of merD, the suspected merTPAB corepressor, could lead to an increase in the mer- Expression contribute in these P. putida :: mer isolates was observed. However, the fact that expression fluctuates strongly between :: mer isolates (see Table 2), again supports the assumption that the transcription into the strains at least partially from exogenous host promoters is triggered at the locations of the mer insertion.

Example 5. Relationship between PMA ^r and Hg ^r and merTPAB expression

The mercury reductase activities of the P. putida :: mer isolates tested in each case were plotted against the resistance of the same strains to PMA or Hg (II) in solid or liquid LP121 media. The results of this analysis are shown in Fig. 2. The data generally have a quadratic function, which shows that the resistance increases with the square (or a portion thereof) of the reductase activity. The resistances measured in liquid LP121 media showed a better correlation to the mer expression than those determined on solid media. In the transformation of all correlations into statistical pairs of numbers, however, only the data obtained from the resistance determinations in liquid PMA-containing LP121 media and the group data fell into an interval of 95% certainty ( FIG. 2). While the mercury resistance is generally correlated with the mer expression under the conditions used here, the resistance to PMA showed the most direct relationship to the mer expression when measured in liquid minimal media.

In sharp contrast to these results, studies in E. coli have shown that with increasing dose of the mer gene, no increase in Hg ^r can be shown; resistance showed no correlation with the values for mer expression (23, 25). Although the mercury reductase activities were found to increase with the copy number of mer in E. coli, no simultaneous increase in Hg ^{r of} the whole cells (23, 25) was evident at these increases. Further investigations showed that Hg ^{r was} limited by the Hg (II) transport into the whole cells; According to these results, the transport reached a saturation point (31). Because MerT, one of the transport proteins, is likely to be an integral protein of the inner membrane (15, 21, 25), it has been suggested that a limitation on the availability of insert types for MerT in the membrane for the observed lack of correlation between Hg ^r and the mer expression could be responsible (23, 31). A limited expression of merPT or energetic considerations regarding the transport were also offered as explanations for the limitation of Hg ^{r in} spite of an increasing number of mer copies in E. coli (31).

The Hg ^r of E. coli strains examined in the studies cited above was determined either on complex and / or solidified media. As expected, sulfhydryl groups in complex media should bind a certain proportion of the available Hg (II). The real concentration of Hg (II) to which the cells are exposed on agar plates is also almost certainly below the expected concentration due to the asymmetrical physiochemical conditions of cells growing on a solid substrate. Differences in Hg ^r between different strains can be weakened under these circumstances, especially if only slight increases in the gene dose are examined. These considerations could also explain the decrease in proportionality observed between the Hg ^r / pMA ^r and the mer expression of P. putida :: mer isolates on solidified media ( Fig. 2). Also, since Hg (II) is lost from liquid media faster than PMA due to its volatility, a better correlation between resistance and expression in PMA-containing liquid media could be achieved, which was observed in the present case ( FIG. 2).

When using P. putida as a genetic background according to the invention found that the hyperexpression of mer conferred increased mercury resistance. Similar attempts in E. coli were unsuccessful, but showed the transport limits tations in this organism (31). Differences in Membrane composition due to genetic divergence these two organisms and / or the composition of the media could explain the diversity of these results. If the membrane capacity for mer-encoded transport proteins in P. putida is significantly increased, this could be the observed direct correlation of mer expression with resistance to Have consequence.  

Example 6. Determination of growth rates in the presence of mercury

Growth curves for the parental, Hg sensitive (Hg ^s ) strains P. putida KT2442 and F1 and their KT2442 :: mer-73, F1 :: mer-1-1 and KT2442-Tn501-132 Hg ^r derivatives were measured, to determine the effect of merTPAB hyperexpression on the growth rate of the culture.

Hyperexpressing merTPAB strains showed no significant effect of Hg exposure on their growth rate when inoculated in HgCl ₂ -containing LP121 media, while both parental P. putida and the Tn501 derivative could not grow under identical conditions ( Fig. 3 ). Since merTPAB expression has been shown to be constitutive, survival of these strains could be expected upon initial mercury exposure. However, induction with Hg is required for the Tn501-containing strain so that the mer operon encoded by Tn501 can be expressed (39); if the cells are exposed to toxic concentrations of Hg (II), this in all likelihood prevents cell growth before the mer induction starts, at the same time simulating an Hg ^s phenotype ( FIG. 3).

The addition of HgCl ₂ to the midlog phase also had no significant effect on the growth rate of :: mer-73 or -1-1, while the parental Hg ^s control cultures showed an immediate standstill of cell growth and an induced P. putida KT2442 : Tn501-132 culture had a slightly increased growth rate ( Fig. 3).

Previous growth studies with the inducible mer-operon of wild type pDU1358 with a broad spectrum of activity in E. coli showed that cultures treated with Hg (II) or PMA concentrations below the toxic range grow at higher rates than those which later have toxic concentrations cations of these compounds were exposed (11). P. putida :: mer strains containing the altered mer system derived from pDU1358, on the other hand, showed a growth rate as high as that of untreated controls even when they were treated with toxic Hg (II) concentrations ( FIG. 3 ).

Example 7. Verification of a stable merTPAB transposition

KT2442 :: mer isolates (-67, -73, -2-4, -2-49, -1-121) and F1 :: mer isolates (-1-1, -1-7, -1-22, -10, -13) from P. putida were analyzed by Southern hybridization (37) to determine the Ensure transposition of the mer operon and uniqueness of the respective transposition events.

Purified pUTHg DNA was digested with SfiI, whereby a 3.2 kB fragment is generated, excluding the shortened 1550 sequences encoded for the merTPAB genes (12). Genomic DNA isolated from the P. putida: mer isolates was cut in the same way with SfiI or EcoRI, for which there is one in the mobilized mini-Tn5-mer-TPAB construct single interface there (12). Genomic DNA of the parent Chen strains P. putida KT2442 and F1 were also identified with SfiI digested. P. putida KT2442 :: Tn501-132 genomic DNA digested with AvaI, resulting in a 2.7 kB fragment that contains the Tn501 merTPAD genes (3, 4, 21).

The entire digested DNA was separated individually using either the iso of the 3.2 kB pUTHg fragment that was used for merTPAB encoded, or pooled with pGP704 as the probe pUTHg vector sequences (minus mini-Tn5 and tnp *) contains (12, 20).

The results showed that when combined with the mer sequence, a 3.2 kb hybridizing fragment was present in all SfiI digested P. putida :: mer samples, which corresponds to the pUTHg-mer DNA generated by SfiI, which shows that merTPAB is actually encoded by these isolates ( Fig. 4). Higher molecular weight fragments from these digestions that show less hybridization are likely to be the result of incomplete SfiI digestion of the hybridized DNA ( Fig. 4). The DNA containing KT2442-Tn501, when combined with merTPAB as a probe, showed hybridization of a fragment corresponding to the predicted size of the Tn501-merTPAD DNA (3, 4, 21). As expected, the parental strains KT2442 and F1 showed no hybridization with the mer probe ( FIG. 4).

Hybridization of the mer probe with P. putida :: mer DNA digested with EcoRI resulted in two hybridizing fragments per isolate; all fragments differed in their mobility (KT2442 derivatives are shown in Fig. 4). As a result, the mer transposition occurred only once in these strains and resulted in the integration into a single host sequence which occurred only once.

When trying to hybridize the same set of DNA with the mer cloning vector pGP704 showed only the pUTHg control positive hybridization (data not shown). The existence from merTPAB in the P. putida :: mer descendants is not the result of a cointegration, since no vector sequences are observed were respected, but shows real transposition events.

Example 8. Growth and dioxygenase activity of P. putida F1 :: mer isolates on PMA

Since P. putida F1 :: mer isolates both by the transposon coded organo mercury lyase as well as one by their Chromosomes include specified catabolism for benzene (10), they were examined to determine whether they had both Hg (II) split off from PMA as well as the formed benzene group as could use only carbon and energy sources. Des half the ability of 21 P. putida F1 :: mer- Isolates analyzed on M63 agar with 160 µg / ml PMA as one ziger carbon source to grow. Emerging colonies were the presence of a color test with 0.5 M catechol activity of one of the enzymes involved in benzene degradation, Catechol-2,3-dioxygenase (9).  

Both controls, P. putida F1 and P. putida KT2442 :: mer73, were unable to grow on PMA (Table 3). P. putida F1 has no Hg ^r with a broad spectrum of activity and is therefore sensitive to PMA (PMA ^s ) ( Fig. 1). In addition, PMA may not be available as a carbon source for P. putida F1 because this organism does not code for an organomercury lyase and its aromatic catabolism should not recognize PMA as a substrate. P. putida KT2442 :: mer-73, on the other hand, codes for PMA ^r ( FIG. 1) and can therefore form benzene, but cannot grow on this substrate because it lacks a benzene degradation pathway (Table 3).

Nine of the 21 P. putida F1 :: mer strains tested were found M63-PMA media grow and show dioxygenase-Ab on them activity (strains, for the mercury reductase determinations were shown in Table 2). The There were no open differences in the growth of the strains on PMA visible correlation to the measured values for the merA Expression on (compare Tables 2 and 3). Imputed comparable reductase values between :: mer strains are an indication for corresponding lyase values (since both merA as well as merB in all these strains from the same pro motor can be expressed), then the fact that both mer expressors with high values as well as those with low values growing values on PMA, not by referring to corresponding corresponding values of the lyase activity can be explained: Activity could not have been too low for growth to allow on the benzene formed. That very question does not seem to be clear from the available data and requires further investigation.

The apparent incompatibility between the observed growth to 160 µg / ml PMA in M63 minimal medium and a maximum PMA ^r of 80 µg / ml on LP121 can best be explained by a connection with the respective medium composition. Since LP121 offers the cells phosphate-limiting conditions (100 µM), they are probably under stress and therefore show a lower PMA ^r than under the phosphate-unlimited conditions of the M63 medium. In any case, with the help of progeny that show growth on PMA, the activities of Hg ^r with a broad spectrum of activity can be combined with the endogenous degradation capabilities of P. putida F1 (10) and a new catabolic pathway can be created.

The present invention represents Orga for the first time nisms are available in which the overexpression of for the Mercury detoxification required genes with a clear Associated with increased mercury resistance.

Overexpression in E. coli is already inso succeeded far as the number of copies of the mer gene were increased could; however, this had no increase in mercury Resistance. It could be shown that the Hg (II) transport under these conditions in E. coli limitie rend was. One of the transport proteins is in the cell membrane inserted integrally, and probably the number of for Hg (II) transport proteins limited available insertion sites. Therefore, over-expressing E. coli strains Hg (II) under high concentrations, it does not enter quickly enough port (and then reduce) to yourself in front of the toxi protect against the cation.

According to the invention, P. putida was now an organism provided in which the overexpression and the resistance ge are coupled. The reason for the coupling could be that the P. putida membranes have more insertion sites for that provide mer transport proteins than that of E. coli, so that the transport into the cell and thus the Hg (II) reduction rate can be increased.

A second important advantage of the systems according to the invention is the constitutive expression of the proteins. The cells Therefore do not die before the Hg (II) reduction indu is decorated as the wild-type isolates do. Even in an order given that with low concentrations of Hg (II) cont  are organisms with constitutive expression consider: While such concentrations are not sufficient are sufficient to induce protein syn in the wild types effecting these and mercury reduction reduce the strains according to the invention also Hg (II) in high dilutions. This is of great advantage since it has been shown that Hg, even if it is in highly diluted and therefore (still) harmless concentrations in the area are high organisms accumulated and thus long-term toxic and is stressful.

Living material

P. putida KT2442; Gene, 16 (1981) 237-247
P. putida F1; J. Biol. Chem., 1989, 264, 14940-14946
E. coli SM10 lambda-pir (pUT-Hg); see. J. Bacteriol., 172 (1990) 6568-6572

literature

1. Bauchop, T., and SR Eidsen. 1960. The growth of microorganisms in relation to their energy supply. J. Gen. Microbiol. 23: 457-469.
2. Begley, TP, AE Walts, and CT Walsh. 1986. Bacterial organomercurial lyase: overproduction, isolation an characterization. Biochem. 25: 7186-7192.
3. Brown, NL, SJ Ford, RD Pridmore, and DC Fritzinger. 1983. Nucleotide sequence of a gene from the Pseudomonas transposon Tn501 encoding mercuric reductase. Biochem. 22: 4089-4095.
4. Brown, NL, TK Misra, JN Winnie, A. Schmidt, M. Seiff, and S. Silver. 1986. The nucleotide sequence of the mercuric resistance operons of plasmid R100 and transposon Tn501: further evidence for mer genes which enhance the activity of the mercuric ion detoxification system. Mol. Gen. Genet.
202: 143-151.
5. Boyer, HW, and D. Roullard-Dussoix. 1969. A complementation analysis of the restriction and modification of DNA in Escherichia coli. J. Molec. Biol. 41: 459-472.
6. Figurski, D., and DR Helinski. 1979. Replication of an origin-containing derivative of plasmid RK2 dependent on a plasmid function provided in trans. Proc. Natl. Acad. Sci. USA 76: 1648-1652.
7. Foster, TJ, and F. Ginnity. 1985. Some mercurial resistance plasmids from different incompatibility groups specify merR regulatory functions that both repress and induce the mer operon of plasmmid R100. J. Bacteriol. 162: 773-776.
8. Fox, B., and CT Walsh. 1982. Mercuric reductase. Purification and characterization of a transposon-encoded flavoprotein containing an oxidation reduction active disulfide. J. Biol. Chem. 257: 2498-2503.
9. Franklin, FCH, M. Bagdasarian, MM Bagdasarian, and KN Timmis. 1981. Molecular and functional analysis of the TOL plasmid pWW0 from Pseudomonas putida and cloning of genes of the entire regulated aromatic ring meta cleavage pathway. Proc.Natl Acad Sci. 78: 7458-7462.
9a. Frischmuth, A., 1991. Development of a bioreactor for the removal of mercury from aqueous media by an active microbial process. Dissertation FU Braunschweig.
10. Gibson, DT, JR Koch, and RE Kallio. 1968. Oxidative degradation of aromatic hydrocarbons by microorganisms. l. enzymatic formation of catechol from benzene. Biochemistry 7: 2653-2662.
11. Griffin, HG, TJ Foster, S. Silver, and TK Misra. 1987. Cloning and DNA sequence of the mercuric and organomercurial-resistance determinants of plasmid pDU1358. Proc. Natl. Acad. Sci. USA 84: 3112-3116.
12. Herrero, M., V. de Lorenzo, and KN Timmis. 1990. Transposon vectors containing non-antibiotic resistance selection markers of cloning and stable chromosomal insertion of foreign genes in gram-negative bacteria. J. Bacteriol. 172: 6557-6567.
13. Itoh, Y., and D. Haas. 1985. Cloning vectors derived from the Pseudomonas plasmid pVS1. Gene 36: 27-36.
14. Kreuzer, K., C. Pratt, and A. Torriani. 1975. Genetic analysis of regulatory mutants of alkaline phosphatase. Genetics 81: 459-468.
15. Lund, PA, and NL Brown. 1987. Role of the merT and merP gene products of transposion Tn501 in the induction and expression of resistance to mercuric ions. Gene 52: 207-214.
16. Lund, PA, and NL Brown. 1989. Regulation of transcription in Escherichia coli from the more and merR promoters in the transposon Tn501. J. Mol. Biol. 205: 343-353.
17. Mason, RP, and WF Fitzgerald. 1990. Alkyl mercury species in the equatorial Pacific. Nature 347: 457-459.
18. Mermod, N., PR Lehrbach, RH Don, and KN Timmis. 1986. Gene cloning and mainipulation in Pseudomonas, p. 325-355. In JR Sokatch (ed.), The bacteria. vol. 10. Academic Press, Inc., New York.
19. Miller. JH 1972. Experiments in molecular genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
20. Miller, VL, and JJ Mekalanos. 1988. A novel suicide vector and its use in the construction of insertion mutations: osmoregulation of outer membrane proteins and virulence determinants in Vibrio cholerae requires toxR. J. Bacteriol. 170: 2575-2583.
21. Misra. TK, NL Brown, D. Fritzinger, RD Pridsmore, WM Barnes, L. Haberstroh, and S. Silver. 1985. Mercuric ion resistance operons of plasmid R100 and transposon Tn501: the beginning of the operon including the regulatory region and the first two structural genes. Proc. Natl. Acad. Sci. USA 81: 5975-5979.
22. Mukhopadhyay, D., H. Yu, G. Nucifora, and TK Misra. 1991. Purification and functional characterization of MerD. J. Biol. Chem. 266: 18538-18542.
23. Nakahara, H., TG Kinscherf, S. Silver, T. Miki, AM Easton and RH Rownd. 1979. Gene copy number effects in the mer operon plasmid NR1. J. Bacteriol. 138: 284-287.
24. Nakahara, H., S. Silver, T. Miki, and RH Rownd. 1979. Hypersensitivity to Hg (II) and hyperbinding activity associated with cloned fragments of the mercurial resistance operon of plasmid NR1. J. Bacteriol. 140: 161-166.
25. NiBhriain, N., S. Silver, and TJ Foster. 1983. Tn5 insertion mutans in the mercuric ion resistance genes derived from plasmid R100. J. Bacteriol. 155: 690-703.
26. Niebor, E., and DHS Richardson. 1980. The replacement of the nondescript term "heavy metals" by a biologically and chemically significant classification of metal ions. Environ. Pollut. Ser. B 1: 3-26.
27. Nriagu, JO, and JM Pacyna. 1988. Quantitative assessment of worldwide contamination of air, water and soils by trace metals. Nature 333: 134-139.
28. Nucifora, G., L. Chu, S. Silver, and TK Misra. 1989. Mercury operon regulation by the merR gene of the organomercurial system of plasmid pDU1358. J. Bacteriol. 171: 4241-4247.
29. Nucifora, G., S. Silver, and TK Misra. 1989. Down regulation of the mercury resistance operon by the most promoter-distal gene merD. Mol. Gen. Genet. 220: 69-72.
30. OHalloran, T., and C. Walsh. 1987. Metalloregulatory DNA-binding protein encoded by the mergene: isolation and characterization. Science 235: 211-214.
31. Philippidis, GP, LH. Malmberg, WS. Hu, and JL Schottel. 1991. Effect of gene amplification on the mercuric reduction activity of Escherichia coli. Appl. Environ. Microbiol. 57: 3558-3564.
32. Ramos, JL, Wasserfallen, A. Rose, K., and Timmis, KN 1987. Redesigning metabolic routes: manipulation of the TOL plasmid pathway for the catabolism of alkylbenzoates. Science 235: 593-596.
33. Richards, E 1987. Preperation of genomic DNA from bacteria. Miniprep of bacterial genomic DNA, p. 1-2, unit 2.4.1. In FM Ausubel, R. Brent. RE Kingston, DD Moore, JG Seidman, JA Shmith, and K. Struhl (ed.), Current protocols in molecular biology. John Wiley & Sons, Inc. New York.
34. Rojo, F., Pieper, DH, Engesser, KH, Knackmuss, HJ, and Timmis, KN 1987. Assemblage of an ortho cleavage route for the simultaneous degradation of chloro- and methylaramatics. Science 235: 1395-1398.
35. Sambrook, J., EF Fritsch, and T. Maniatis. 1989. Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
36. Schottel, JL 1978. The mercuric and organomercuric detoxifying enzymes from a plasmid-bearing strain of Escherichia coli. J. Biol. Chem. 253: 4311-4349.
37. Southern, EM 1975. Detection of specific DNA sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 98: 503-517.
38. Stanisich, VA, PM Bennett, and MH Richmond. 1977. Characterization of a translocation unit encoding resistance to mercuric ions that occurs on a nonconjugative plasmid in Pseudomonas aeruginosa. J. Bacteriol. 129: 1227-1233.
39. Summers, AO 1986. Organization, expression, and evolution of genes for mercury resistance. Ann. Rev. Microbiol. 40: 607-634.
40. Summers, AO, and L. l Sugarman. 1974. Cell-free mercury (II) - reducing activity in a plasmid-bearing strain of Escherichia coli. J. Bacteriol. 119: 242-249.
41. Tezuka, T., and K. Tonomura. 1978. Purification and properties of a second enzyme catalyzing the splitting of carbon-mercury linkages from mercury-resistant Pseudomonas K-62. J. Bacteriol. 135: 138-143.
42. Timmis, KN 1990. Genetic construction of noval metabolic pathways: degradation of xenobiotica, p. 69-83. In K. Mosbach (ed.), Colloquium 41: Molecular basis of bacterial metabolism. Springer publishing house Berlin.
43. Young, RA, and RW Davis. 1983 Efficient isolation of genes by using antibody probes. Proc. Natl. Acad. Sci. 80: 1194.

Claims (15)

1. bacterial strain, characterized in that it

• i) is able to constitutively overexpress genes (mer genes) required for mercury reduction,
• ii) is resistant to Hg (II) compounds.

2. Bacterial strain according to claim 1, characterized in that it additionally

• iii) toluene and benzene and toluene and benzene groups of organic substances can degrade.

3. bacterial strain according to claim 1 or 2, characterized records that it is a P. putida strain. 4. bacterial strain according to claim 3, characterized in that it is P. putida F1 or P. putida KT2442. 5. Bacterial strain according to one of claims 1 to 4, characterized characterized in that he has the mer genes in his genomic DNA inserted contains. 6. bacterial strain according to claim 5, characterized in that in the mer genes the regulatory genes or sequences entirely or partially deleted. 7. bacterial strain according to claim 5 or 6, characterized records that the mer genes by transposition of pUTHg in the host DNA has been inserted. 8. P putida KT2442 · pUTHg or P. putida F1 · pUTHg.   P. putida KT 2442:: mer-73, deposited under DSM No. 7 209 or P. putida F1:: mer-1-1 deposited under DSM No. 7 208. 10. A method for producing a bacterial strain according to one of claims 1 to 9 by untargeted transposon muta genesis with a transposon containing mer genes. 11. The method according to claim 10, characterized in that the transposon

• i) has a short ending sequence,
• ii) is transposed only once into the acceptor or recipient and remains stably integrated there,
• iii) has no mer regulatory genes.

12. The method according to claim 10 or 11, characterized in net that a transposon-containing plasmid from an E. coli Donor, e.g. B. SM10 λ-pir, by mobilization with a bipa profitable filter mating technology in the appropriate acceptor is transferred. 13. The method according to any one of claims 10 to 12, characterized characterized in that the conjugation products are based on mercury Resistance can be selected. 14. The method according to claim 13, characterized in that selection by screening the isolates with a me dium takes place, which contains phenylmercury acetate. 15. The method according to claim 14, characterized in that screening of isolates with increasing concentrations of Phe nylmercury in the medium.